Devices, solutions and methods for sample collection

ABSTRACT

The disclosure relates to devices, solutions and methods for collecting and processing samples of bodily fluids containing cells (as well as embodiments for the collection, and processing and/or analysis of other fluids including toxic and/or hazardous substances/fluids). In addition, the disclosure relates generally to function genomic studies and to the isolation and preservation of cells from saliva and other bodily fluids (e.g., urine), for cellular analysis. With respect to devices for collection of bodily fluids, some embodiments include two mating bodies, a cap and a tube (for example), where, in some embodiments, the cap includes a closed interior space for holding a sample preservative solution and mates with the tube to constitute the (closed) sample collection device. Upon mating, the preservation solution flows into the closed interior space to preserve cells in the bodily fluid. The tube is configured to receive a donor sample of bodily fluid (e.g., saliva, urine), which can then be subjected to processing to extract a plurality of cells. The plurality of cells can be further processed to isolate one and/or another cell type therefrom. The plurality of cells, as well as the isolated cell type(s), can be analyzed for functional genomic and epigenetic studies, as well as biomarker discovery.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/023,772, filed Jun. 29, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/227,693, filed Aug. 3, 2016, which is acontinuation of U.S. patent application Ser. No. 14/127,832, filed Dec.19, 2013, now U.S. Pat. No. 9,442,046, which is a 35 U.S.C. § 371national stage entry of PCT/US2012/043176, filed Jun. 19, 2012, andclaims priority under 35 USC § 119(e) to U.S. provisional patentapplication nos. 61/498,584, filed Jun. 19, 2011, 61/598,601, filed Feb.14, 2012, and 61/598,618, filed Feb. 14, 2012. Each disclosure of theforegoing is herein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates to devices, solutions and methods for collectingsamples of bodily fluids or other substances, including hazardous and/ortoxic substances, and in particular, a naturally expressed bodily fluid(e.g., saliva, urine). In addition, the disclosure relates generally tofunctional genomics and to the isolation and preservation of cells fromsuch bodily fluids, for studies in any of: functional genomic andepigenetic studies, and biomarker discovery (for example).

BACKGROUND

Personalized medicine is the customization of treatment to an individualas opposed to the one treatment-for-all model. Personalized medicineinvolves categorizing a patient based on his or her physical conditionand designing an optimal healthcare solution exclusively for thatcategory. The progression of personalized medicine is dependent on thediscovery, validation, and commercialization of biomarkers to stratifypopulations for treatment and for the development of diagnostics forscreening and early detection.

Epigenetic research has come to the forefront of medical research and isimplicated in the etiology of a number of physical and mental illnessesincluding: cancer, obesity, diabetes, schizophrenia, and Alzheimer'sdisease (Alika et al., 2010; Grant et al. 2010; McGowen et al., 2009;McGowen and Szyf, 2010; Plazas-Mayorca and Vrana, 2011; and Portela andEsteller, 2010). In addition, Epigenetics may hold particular promise inthe many scientific and medical areas including but not limited to:cancer, diabetes, drug integrations, drug effectiveness, childhoodaggression, suicidal behaviors, aging, inflammation, pain, obesity,schizophrenia, and other mental illnesses (Abdolmaleky et al., 2005;Costa et al., 2003; Iwamoto & Kato, 2009; Kuratomi et al., 2007; McGowan& Kato, 2007; McGowen and Szyf, 2010; Peedicayil, 2007; Petronis et al.,1999; McGowen and Szyf, 2010; Plazas-{hacek over ( )} Mayorca and Vrana,2011; and Zawia et al., 2009).

A major challenge in the field includes the identification of anappropriate source material for home-based sample collection that isadequate for large-scale epigenetic research includingwhole-genome-analysis studies. Epigenetics may be the key forunderstanding the mechanisms of gene-environment interactions as growingevidence suggests that epigenetic mechanisms may provide a molecularmemory of environmental experiences (Ho, 2010; Kappeler and Meaney,2010; McGowen et al., 2009, McGowen and Szyf, 2010; Portela andEsteller, 2010; Richards, 2008; Russo et al., 2010; Tsai et al, 2010;and Vlaanderen et al., 2010). Preliminary data from some humans suggestthat distinct methylation patterns in peripheral blood cells areassociated with social behaviors including: childhood aggression,suicidal behaviors, and ageing (Kappeler and Meaney, 2010; McGowen etal., 2009; McGowen and Szyf, 2010; Portela and Esteller, 2010; Russo etal., 2010, Tierling et al., 2010; Tsai et al, 2010; and Zhang et al.,2011).

Due at least in part to the heterogeneous nature of human disease,particularly mental illness, and the complex interaction of contributingetiological factors, studies require large sample sizes to providereliable and significant effects. However, current research options forsample collection for epigenetic studies do not meet this requirement of“large sample sizes.” The need for large sample sizes for studies isalso true in order to produce significant effects in regards to studyinghuman-environment interactions as these interactions are also of a verycomplex nature with many contributing factors. The ability to performlarge-scale “population sized” (subject samples numbering in at leastthe hundreds to thousands) epigenetic research can introduce a newunderstanding of human-environment interaction and facilitate thecompletion of longitudinal studies facilitating the development ofepigenetic-based screening diagnostics crucial to the progression ofmodern medicine. This epigenetic research may lead to a newunderstanding of how the environment affects our epigenome and how thisrelates to a person's health outcome, which may further lead to thedevelopment of preventative interventions for individuals who areconsidered high-risk and diagnostics for these health disparitiesincluding, but not limited to, diagnosis.

Some epigenetic studies attempting to quantify environmental and othercomplex interactions in human populations use blood as the sourcematerial for experimentation. Blood can restrict the researcher'sability to conduct large population-sized studies as it:

-   -   1. generally requires medical supervision,    -   2. involves invasive procedures for collection,    -   3. carries stigma that limits participation, and    -   4. is expensive to collect and ship.

Naturally expressed bodily fluids, e.g., saliva and urine, can be anadditional or alternative appropriate source material for home-basedsample collection as they:

-   -   1. do not require invasive techniques,    -   2. do not have the same stigma as blood,    -   3. do not require professional supervision, and    -   4. can be inexpensive to collect.

In addition, at least saliva has been shown to contain white blood cells(Dos-Santos et al., 2009). The use of bodily fluids, e.g., saliva,urine, may enable large-scale “population-sized” epigenetic research. Inaddition, home-base sample collection of saliva, or urine, may allow fora much wider range of research options available as it can greatlyincrease participant numbers and samples can be more easily shipped bythe subjects from anywhere in the world. For example, the ability tomore easily ship samples from anywhere in the world can be particularlyuseful when samples are from countries that do not have laboratoryinfrastructure.

An organism's genome is a fixed sequence that contains its hereditaryinformation and is the same in every cell of an organism. An organism'sepigenome, by contrast, varies between cell types and changes over theorganism's lifetime. Thus, epigenetic studies may include a single celltype as the source of sample material to control for these differences(Johnson and Tricker, 2010; Lister et al., 2009; and Rangwala et al.,2006). For example, human saliva contains numerous cell types, includingepithelial cells, cells normally found in the blood (i.e., T-cells andB-cells), bacteria and debris (Dos-Santos et al., 2009 and Viet andSchmidt, 2008). The cells in saliva that are the most important toprofile epigenetically are those that come from the blood stream, asthese cells carry epigenetic information from the entire body (Kappelerand Meaney, 2010; McGowen and Szyf, 2010; McGowen and Szyf, 2010;Righini et al, 2007; Rosas et al., 2011, Vlaanderen et al., 2010 andZhang et al., 2011).

Additionally, it may not be practical to use whole saliva DNA as thecells in saliva that are not found in the blood, such as epithelialcells, which make up the vast majority of cells in saliva (Dos-Santos etal., 2009) have the ability to “mask” the epigenetic effects seen inT-cells (cells that originated in the blood) by dampening the effect ofthe minority of cells (Dos Santos et al., 2009, Lister et al., 2009; andTierling et al., 2010). To address these concerns AboGen developed amethod to separate and extract the different cell types found in bodilyfluids such as saliva by taking advantage of cell-specific markers andisolation techniques (e.g., magnetic). This method uses practicalamounts of bodily fluids, such as saliva, to yield enriched cells thatcan be used for downstream biological applications including large-scalefunctional genomic studies (example epigenomic studies). For example,saliva sample processing technology allows collected samples to beprocessed into single cell types and have their epigenomes profiled.

Furthermore, saliva (and other bodily fluids) can present challengeswith cell isolation as a source material for blood cells in respect todownstream experimentation for reasons such as:

-   -   1. Blood is a transporter fluid while saliva is a digestive        fluid that can be rich in proteases, enzymes and secreted        substances and urine is a excretory fluid consisting of unwanted        waste products.    -   2. Some fluids can have a wide pH range and some of the pH        values reported, such as for saliva, would result in death if        blood reached that pH (saliva is 6.2-7.4; urine is 4.5-8; blood        is 7.35-7.45).    -   3. Some fluids contain more bacteria than blood.    -   4. Some fluids contain non-cellular material that varies between        individuals and interferes with cell isolation.    -   5. Some fluids include blood cells, such as T-cells, which can        be abundant in blood, but may be rare in other naturally        expressed bodily fluids, such as saliva or urine, and are vastly        outnumbered by other cell types, such as epithelial cells,        unlike in blood.    -   6. The subset of lymphocyte cells in some bodily fluids, such as        saliva, greatly differs from the population of those cell types        in blood. For example, only CD4+CD8− T-cells are reported to be        found in saliva.    -   7. Some fluids are produced each day, such as saliva at about a        rate of 0.5-1.5 liters per day per person.

Therefore, there is a need for new methods for isolating rare cells(i.e., T-cells) from saliva and other naturally expressed bodily fluids.

For collecting saliva samples from a large population of people(example: functional genomic studies) who are widely geographicallydispersed, several requirements may need to be met for an optimal samplecollection device. For example, it may be beneficial to have the samplecollection device securely house a toxic preservative solution in aclosed chamber. Additionally, the sample collection device may be ableto be sent to a donor with the toxic solution safely enclosed. Thesample collection device may also allow easy and safe collection of adonor specimen, such as human saliva or urine, with no risk of exposureof the donor to the toxic solution. Furthermore, the sample collectiondevice may allow the donor to safely mix the toxic solution and thespecimen (for preservation of the specimen) with no risk of exposure ofthe donor to neither the toxic solution nor any other hazard. The samplecollection device may also allow the donor to send the sample collectiondevice to a laboratory for processing generally “as-is” after securelyclosing the sample collection device. Finally, the sample collectiondevice may further allow a laboratory technician to receive the samplecollection device and safely open it for processing with generally norisk of exposure to any hazards.

Some currently available sample collection devices include, for example,U.S. Pat. No. 7,482,116 which describes a device that utilizesdisassociating a barrier to allow fluid communication between a cavityholding the donor sample and a solution, however, embodiments includedin the patent are limited to the use of sharp extruding objects and thinpierceable membranes. The thin pierceable membranes can represent asafety hazard to the sample donor as any wrong manipulation (such aswith a finger nail) can lead to piercing of the membrane and release ofthe solution. US patent publication no. 2009/0216213 A1 claims a devicethat utilizes a pierceable membrane to establish fluid communicationbetween a cavity containing a solution and the donor sample. This canrepresent a safety hazard to the sample donor as any wrong manipulationcan lead to piercing the membrane and exposing the solution. The devicealso requires exchange of the cap prior to sending the sample to the enduser. This can represent a safety hazard as it may expose the sampledonor to the potentially toxic solution. Therefore, there is a need forsafer and easier to use sample collection devices.

Additionally, the purification process requires cells to maintain theirantigen profiles and the epigenomic profiling requires that theirepigenome be maintained. To this end, it is necessary to treat the cellsin such a way that they are able to generally maintain these features.Currently available treatments generally do not meet this need. Forexample, U.S. Pat. Nos. 7,267,980 and 7,749,757 disclose solutionscontaining lysine, glycine and formaldehyde for stabilizing cells fromblood. However, those solutions will not protect cells from proteasesfound in some bodily fluids, such as saliva. Therefore, there is a needfor new solutions and methods that will preserve the antigenicity andepigenome of cells in other bodily fluids, such as saliva.

SUMMARY OF THE DISCLOSURE

Embodiments of the disclosure provide safer and easy to use samplecollection devices for naturally expressed bodily fluids (for example),as well as solutions and methods for preserving cells of samplescollected, and additionally, methods for isolating specific cells eithercollected and/or preserved. Such isolated cells (and even non-isolatedcollected cells), can then be analyzed for studies in any of: functionalgenomic and epigenetic studies, and biomarker discovery (for example).

The sample collection devices according to the present disclosureprovide several advantages over currently available sample collectiondevices. For example, in some embodiments, the sample collection devicesuse a minimum amount of parts and do not require removal or exchange ofa piece or an object thereof. In some embodiments, the sample collectiondevices do not require any additional manipulation by the sample donorapart from depositing the sample in the sample collection device andclosing the sample collection device. In some embodiments, use of thesample collection devices provide improved safety for both the sampledonor and the end user, since, for example, sharp objects are notincluded and there is limited to no risk of exposure to toxic solutions(e.g., sample preservative solutions).

In some embodiments of the sample collection device, the samplecollection device can have two main mating bodies, a cap and a tube. Thecap can include a closed cavity holding a preservative solution whichcan mate with the tube to constitute the closed sample collectiondevice. The tube can be configured to receive the donor specimen. Thecap and tube are configured so that when the donor deposits the specimenand closes the tube with the cap, the cavity holding the preservativesolution may be opened to release the preservative solution and allow itto mix with the donor specimen.

In some embodiments, a bodily fluid sample collection device for thecollection of naturally expressed bodily fluids is provided and includesa cap having an outer wall having an engagement member, and an interiorchamber for holding a fluid. The chamber may comprise inner walls whichdefine an interior space and an aperture, where the aperture isconfigured for sealing by a removable blocking member. The blockingmember may include a first coupling member for engaging a correspondingsecond coupling member in a tube, thereby causing removal of theblocking member and opening of the aperture when the cap is coupled tothe tube. The device also includes the tube which includes a containmentwall defining a reservoir for bodily fluid sample collection, anengagement member complementary to the engagement member of the cap, andthe second coupling member.

In some embodiments, one and/or another of the following features may beprovided with a sample collection device:

-   -   the removable blocking member is a disk-shaped member which        threadably engages the aperture;    -   the first coupling member comprises an indentation disposed        centrally in the bottom of the blocking member and the second        coupling member is disposed centrally within the tube;    -   the first coupling member comprises a recess disposed        eccentrically in the bottom of the blocking member and the        second coupling member is disposed eccentrically within the        tube;    -   the removable blocking member comprises an annular member having        threads arranged thereon, where the annular blocking member        substantially covers the aperture, and the inner wall of the cap        includes complementary threads, such that the annular member can        be screwed into the interior space to uncover the aperture;    -   a locking mechanism, to lock the cap to the tube (or lock any        two components together), the locking mechanism may comprise a        wedge and a complementary flange;    -   a sealing mechanism which may comprise a sealing substance        associated with the engagement member of the cap, where upon        coupling the cap to the tube, the sealing substance flows into        at least the engagement member of the cap;    -   tamper-evident means for determining whether the cap has been        opened, which may comprise a ring having a first portion thereof        integral with an open end of the cap, where upon the cap being        coupled to the tube, the ring is positioned adjacent the tube;        as such, in some embodiments, upon the cap being de-coupled from        the tube, the first portion is broken and the ring remains        substantially adjacent the tube; and/or the fluid in the cap        chamber comprises a solution for preserving cells.

In some embodiments, a bodily fluid sample collection device for thecollection of naturally expressed bodily fluid is provided and includesa cap having an interior chamber for holding a fluid and a firstengagement member, and a tube comprising a containment wall defining areservoir for sample collection and a second engagement member forengagement to the first engagement member. In some such embodiments, thecap comprises an outer wall having the first engagement member, thechamber comprises inner walls defining an interior space which holds thefluid, and an aperture, the aperture being configured for sealing by aremovable blocking member. In addition, in some embodiments, theblocking member includes a first coupling member for engaging acorresponding second coupling member of the tube, where upon thecoupling of the cap to the tube, the blocking member is moved and theaperture opens.

In some embodiments, a method for collecting a sample of a naturallyexpressed bodily fluid (or toxic or hazardous fluid) is provided andincludes providing a bodily fluid collection device according to any ofthe disclosed sample collection device embodiments, depositing thebodily fluid into the chamber, and mating the cap and tube together suchthat the corresponding engagement members engage, where the blockingmember moves and the preservation fluid flows into the reservoircontaining the bodily fluid such that cells contained in the bodilyfluid are preserved for analysis. In some such embodiments, furthersteps may include at least one of (with reference to bodily fluids):isolating one or more cell types for a plurality of cell types in thebodily fluid, and analyzing the collected cells.

As one of skill in the art will appreciate, in some embodiments, atleast one of DNA, RNA and proteins can be extracted fromcollected/preserved cells, whether the isolated cells, or non-isolatedcells.

In some embodiments, a kit for the collection of naturally expressedbodily fluids (or toxic and/or hazardous fluids) is provided andcomprises a plurality of sample collection devices according to of thedisclosed sample collection devices.

In addition, the current disclosure relates to functional genomicstudies including epigenetic studies. More particularly, this disclosurealso relates to the isolation of cells from bodily fluids, such assaliva and urine, for these studies. Accordingly, some embodiments ofthe disclosure include methods for preserving the antigenicity andepigenome of cells, and isolating rare cells, including, withoutlimitation T-cells from bodily fluids, such as saliva and urine, aredisclosed herein.

As used herein, the collection of “bodily fluids” generally refers tothe collection of naturally expressed bodily fluids (although someembodiments can be used for collection of intravenous collectionmethods—e.g., blood). Thus, with references to the disclosedembodiments, “bodily fluids” refer to naturally expressed bodily fluidsincluding, for example, saliva and urine.

For example, in some embodiments, a solution for preserving cells inbodily fluids, such as saliva and urine, is provided for furtherseparation into cell types and downstream analysis that allows for thecells in saliva to retain their antigenicity and cellular architectureduring storage. The solution can contain at least one chemical fixingagent, such as but not limited to paraformaldehyde, and at least oneprotease inhibitor. In some embodiments, the solution may furthercontain, for example, one or more of: at least one antimicrobial agent,serum proteins from human and/or other animal species. The solution maybe buffered at a pH between about 6.4 to about 8.4, and in someembodiments, between about 7.2 to about 7.6.

In some embodiments, a method for preserving cells in one or more bodilyfluids includes contacting collected cells with a solution according toone and/or another embodiment of the present disclosure, which allowsthe cells to retain their antigenicity and epigenome, for example.

In some embodiments, a method for isolating cells from chemically fixedcells collected from a bodily fluid, e.g., saliva or urine, and includescentrifuging the cells to separate, for example, DNA and/or othersoluble material from a pellet of cells, bacteria, and debris, enrichingwhite blood cells from other contents of the pellet, and isolatingspecific cells (e.g., white blood cells) using antibodies conjugated tomagnetic beads targeted to cell specific markers.

In some embodiments, methods for isolating a particular type of cell,for example, a type of white blood cell (e.g., lymphocytes), from one ormore bodily fluids (e.g., saliva and/or urine), and includes one or moreof the following steps (and, depending upon the embodiment, several orall of the following steps): providing a sample of bodily fluidcomprising chemically fixed cells, optionally centrifuging the bodilyfluid sample to obtain a pellet comprising cells, optionallyre-suspending the pellet in a buffer, subjecting the re-suspended pelletto density gradient separation to obtain a layer of a mixture of whiteblood cell types (including lymphocytes), contacting the mixture of celltypes with a solution containing specific binding agents for an epitopefound on a particular type of white blood cell, and separating theparticular type of white blood cell (including lymphocytes) from themixture of white blood cell types.

In some embodiments, the specific binding agents may be magnetic beadscoupled to antibodies specific to an epitope found on a particular typeof white blood cell, and in the separation step may then comprise, forexample, magnetically separating the particular type of white blood cell(including lymphocytes) from the mixture of white blood cell types(though other cell separation techniques are within the scope of thedisclosure).

In some embodiments, the bodily fluid (e.g., saliva, urine) can be mixedwith a chemical fixative solution and the mixture can be removed fromthe pellet. The pellet can then be re-suspended in a buffer. There-suspended pellet may optionally be centrifuged and washed one or moretimes in the buffer. The washed pellet may then be applied to ahydrophilic polysaccharide mixture to form a gradient. This gradient maybe different than that used for blood because the density of the cellsin other bodily fluids (e.g., saliva, urine) after chemical fixation forpreservation can be different due to the different density of thepreserved cells requiring an alteration in the time, temperature, and/ordensity of the gradient for the cells to be processed through thisdensity gradient.

Additionally, in some embodiments, the white blood cells can form alayer in the gradient. The white blood cell layer can be extracted fromthe gradient and placed in another centrifuge tube where it may bewashed in a buffer and re-pelleted to remove the remaining gradientmixture. The pellet may then be re-suspended and incubated in a buffercontaining antibodies that are conjugated to magnetic beads and specificto antigens that are specific for a cell type to be isolated. In someembodiments, the cell type to be isolated is T-cells and the antigen isa T-cell-specific antigen. In some embodiments, the antigen is CD4. There-suspended cells in the buffer can be bound by the antibody andsubjected to a magnetic field that magnetically attracts the cells boundto the antibody-conjugated magnetic beads to the side of the tube.Remaining liquid may then be removed from the tube and the tube iswashed in buffer. Isolated T-cells then remain attracted to the side ofthe tube and are ready for further processing, such as freezing forlater downstream experimentation (for example).

In some embodiments, a method for preserving cells in a naturallyexpressed bodily fluid comprises contacting the bodily fluid with thepreservation solution according to any of the disclosed embodiments.

The devices, solutions and methods of sample collection, preservation,isolation and analysis will be better understood in light of thefollowing drawings, detailed description and claims. Like referencesymbols in the various drawings indicate like elements.

It is worth noting that while some embodiments of the sample collectiondevices disclosed herein are set forth for use with the collection ofbodily fluids, the same also has particular use with the collection ofany other substance, including hazardous and/or toxic fluids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sample collection device comprising a cap and a tubeaccording to some embodiments of the present disclosure.

FIG. 1A is a cross section view taken along line 1A-1A of FIG. 1 andshows the interior chamber of the cap comprising inner walls whichdefine an interior space and an aperture according to some embodimentsof the present disclosure.

FIG. 1B is a cross section view taken along line 1B-1B of FIG. 1A andshows a coupling member centrally positioned within the tube accordingto some embodiments of the present disclosure.

FIG. 2A shows a longitudinal cross section view of a sample collectiondevice in which the cap contains an inner chamber with a removableblocking member that has an eccentrically located coupling feature whichcan mate with a coupling member eccentrically located in the tubeaccording to some embodiments of the present disclosure.

FIG. 2B is a cross section view taken along line 2B-2B of FIG. 2A andshows a coupling member eccentrically positioned within the tubeaccording to some embodiments of the present disclosure.

FIG. 3A shows an embodiment of the cap of the sample collection devicein which the cap contains an inner chamber with a movable annular memberthat can cover an aperture in the inner wall according to someembodiments of the present disclosure.

FIG. 3B shows an embodiment of the sample collection device in which thecap is coupled to the tube and the movable annular member is moved to aposition where it does not cover an aperture in the inner wall accordingto some embodiments of the present disclosure.

FIG. 3C is a top view of the tube shown in FIG. 2B and shows a couplingmember positioned within the tube according to some embodiments of thepresent disclosure.

FIG. 4A shows an embodiment of the sample collection device comprising alocking mechanism disposed within the inside of the cap and the tube,which prevents the cap from being removed by at least the donor afterthe cap has been coupled to the tube according to some embodiments ofthe present disclosure.

FIG. 4B shows the locking mechanism in the sample collection deviceshown in FIG. 4A showing a locked configuration and an unlockedconfiguration according to some embodiments of the present disclosure.

FIG. 5A shows a sample collection device comprising a locking mechanismdisposed on an outer surface of the cap and tube, which prevents the capfrom being removed by at least the donor after the cap has been coupledto the tube according to some embodiments of the present disclosure.

FIG. 5B shows the locking mechanism in the sample collection deviceshown in FIG. 5A showing a locked configuration and an unlockedconfiguration according to some embodiments of the present disclosure.

FIG. 6 shows a sample collection device further including a sealedcavity containing a sealing solution that is released into theengagement features of the cap and tube when the cap is coupled to thetube, which prevents the cap from being removed by at least the donorafter the cap has been coupled to the tube according to some embodimentsof the present disclosure.

FIG. 7A shows a “tamper-evident” cap, in which an annular member at thebottom of the cap can break away from the cap if the cap has beenremoved after having been rotated/screwed onto the tube according tosome embodiments of the present disclosure.

FIG. 7B shows the “tamper-evident” cap shown in FIG. 7A showing theannular member broken away from the cap according to some embodiments ofthe present disclosure.

FIG. 8 shows the time course of DNA yield in samples stored in chemicalfixative solution at room temperature after 0, 1, 2 and 7 days, as wellas DNA extracted from T-cells from each sample according to someembodiments of the present disclosure.

FIG. 9 is a chart illustrating the relative yield of extracted T-cellsper ml of starting material (e.g., sample of bodily fluid), as comparedto a yield of T-cells from blood.

FIG. 10 shows a saliva dose curve of micrograms of isolated T-cell DNAper ml of saliva according to some embodiments of the presentdisclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments of the present disclosure include devices, solutions andmethods for the collection of samples, such as bodily fluids, as well asmethods for isolating one or more cell types from collected cells(chemically fixed or otherwise). For example, in some embodiments, thesample collection devices provide several advantages over currentlyavailable sample collection devices, and in addition, the samplecollection devices according to some embodiments use a minimum amount ofparts and the devices do not require removal or exchange of a piece oran object. Furthermore, in some embodiments, the sample collectiondevices may generally not require additional manipulation by the sampledonor apart from depositing the sample and closing the collectiondevice. The sample collection devices according to some embodimentsinclude improved safety of use for both sample donors and end users due,at least in part, to the elimination of sharp objects and limited riskof exposure to toxic solutions, as will be described in greater detailbelow.

In some embodiments, methods for the preservation and isolation of cellsfrom bodily fluids for functional genomic and epigenetic studies, aswell as biomarker discovery, are provided. Additionally, this disclosureprovides devices, solutions and methods for isolating rare preservedcells, such as T-cells, from bodily fluids (i.e., saliva, urine), aswill also be described in greater detail below.

Some embodiments of the sample collection device may include two matingbodies, such as a cap and a tube. In some embodiments, the cap mayinclude a closed cavity, such as an interior space, for holding apreservative solution (which may be toxic) for mating with the tube toconstitute a closed sample collection device. The tube may be configuredto receive a donor specimen, such as one or more bodily fluids (e.g.,saliva, urine). In some embodiments, the cap and/or tube may beconfigured so that when the donor deposits the specimen and closes thetube with the cap, the cavity in the cap, which may be holding thepreservative solution, can be opened to release the preservativesolution and allow it to mix with the donor specimen.

One of skill in the art will appreciate that with respect to someembodiments of the collection device described herein, such may be usedin combination with accessories that ease specimen deposit within thecollection device, including, for example, mouth adapters for salivacollection, funnels and hoses for urine collection, and the like.

In some embodiments, the sample collection device may comprise a caphaving an outer wall with interior threads. Additionally, the samplecollection device may include an interior chamber for holding a fluidwith the chamber comprising walls defining an interior space and athreaded aperture in the wall. The aperture in the wall may be sealed bya threadably removable blocking member, where the blocking member mayinclude engaging members for engaging a coupling member in a tube,thereby causing the blocking member to be removed and the aperture toopen when the cap is threaded onto the tube (in some embodiments). Insome embodiments, the sample collection device may further include atube comprising a containment wall defining a lumen or reservoir forsample collection, exterior threads complementary to the interiorthreads of the outer wall of the cap, and a coupling member that has ashape which is complementary to the engaging member in the cap.

In some embodiments, the threadably removable blocking member can be adisk-shaped member that is at least one of pushed, rotated, screwed,threaded, and/or mated into the aperture of the inner chamber and can beat least one of pushed, rotated, screwed, threaded, and/or mated intothe chamber by interaction between the engaging member of the cap andthe coupling member of the tube when the cap is rotated or screwed ontothe tube. The engaging member can be either centrally or eccentricallylocated in the disk-shaped member, with the coupling member being atleast one of centrally or eccentrically located in the tube,respectively.

The terms push, rotate, screw, mate as well as thread, couple, andattach, as well as any corresponding tenses and plurals thereof (asadditionally including the term “feature(s)), disclosed herein,correspond to structure (well known to those of skill in the art) forconnection (either permanent or temporary) of two (or more) components(e.g., “screw means” “mating means”, “coupling feature”, “engagementfeature”). For example, with respect to “pushing”, such means can covera “snap-fit” type of structure; rotation means can cover means in whicha protruding member is received by a corresponding recess when onecomponent is rotated relative to another. “Screwed” and “threadably”covers helical threaded engagement and the like. Thus, use of any ofthese terms (or tenses thereof) can also cover such connection with anysuch means or the equivalents thereof.

In some embodiments, a threadably movable annular member may not fitinto the aperture, but rather covers the aperture from the outside ofthe inner chamber. In such embodiments, the annular member can haveinterior threads complementary to threads on the outside of the innerchamber or interior space. Interaction between the coupling features ofthe annular blocking member and the coupling member of the tube cancause the annular member to be screwed up the outside of the innerchamber, away from the aperture.

In some embodiments, the sample collection device may further includelocking or sealing means, such that the cap cannot be removed from thetube by the donor once the cap has been connected or screwed onto thetube, such as by the donor. Suitable locking members can include a wedgeon the cap and a matching flange on the tube or visa-versa. The wedgeand flange can either be on the inside of the cap and tube, or on theoutside of the cap and tube. Suitable sealing means include a sealedcavity containing a sealing solution, such as a glue, wherein thesealing solution is released when the cap is pushed, rotated or screwedonto the tube and thereafter cures in order to prevent disengagementbetween the cap and tube. In some embodiments, the sealing solution maybe a two-component glue, such as an epoxy, with one component beingsealed into the cap, and the other component sealed into the tube, suchthat the two components mix within the threads when the cap is screwedonto the tube. In other embodiments, the sealing solution can be asingle component, such as a cyanoacrylate-based glue, which can be in asealed cavity in the cap or tube, such that the sealing solution isreleased into the threads when the cap is screwed onto the tube. In someembodiments, the sealing solution can cure soon after engagement betweenthe cap and tube such that disengagement between the tube and cap by theuser can be generally prevented.

Alternatively, or in addition, some embodiments may further include anannular member at the base of the cap that is partially secured to thecap, such that removal of the cap after it has been screwed onto thetube breaks the bond between the cap and the annular member, therebyindicating that the tube has been opened. This “tamper-evident”embodiment is similar to those used to attach a cap to a soda bottle.

The sample collection devices according to some embodiments can be madeof any suitable plastic, such as polypropylene, polystyrene andpolycarbonate. The dimensions of the device can be modified to suit thespecific processing the sample will be subjected to. In certainembodiments, typical dimensions include the following. For the innerchamber of the cap, the volume is from about 3 ml to about 10 ml,typically about 6 ml. For the lumen of the tube, the volume is fromabout 15 ml to about 50 ml, typically about 25 ml. Other volumes arewithin the scope of some embodiments of the present disclosure.

With respect to the figures, FIG. 1 is an illustration of an embodimentof a sample collection device 10 comprising a cap 12 and a tube 14. Thetube can be configured for collection of one or more sample bodilyfluids, and the cap can be configured for storing one or morepreservation fluids. Additionally, the cap 12 and tube 14 can beconfigured to securely mate with one another in order to provide asecure containment of at least the sample bodily fluids for storing andshipping. Furthermore, the mechanism by which may be implemented in thesample collection device 10 for securely mating the cap 12 and the tube14 may prevent disengagement between the cap 12 and the tube 14. Onebenefit of preventing disengagement between the cap 12 and the tube 14is that it can prevent at least, for example, contamination of thesample contained in the tube and exposure of any preservation solutions(which may be toxic) to the sample donor, such as those contained in thecap 12.

FIG. 1A shows an example interior chamber 16 of the cap 12 which may bedefined by at least one outer wall 24 and at least one inner wall 18according to some embodiments. The at least one inner wall 18 mayfurther define an interior space 20 and an aperture 22. In addition, theouter wall 24 may include one or more cap engagement features 34 alongat least one side of the outer wall 24 for engaging the tube 14. Forexample, and shown in FIG. 1A, an inside surface 26 of the outer wall 24can include one or more cap engagement features 34, such as threads, forengaging and mating with one or more complimentary tube engagementfeatures 38, such as threads, associated with the tube 14. The tube 14may be comprised of at least one containment wall 32 which may define areservoir 40 for collecting and storing sample body fluids, such assaliva or urine. An outer surface 30 of the containment wall 32 mayinclude the one or more tube engagement features 28, such as threads.

The cap 12 may further include an aperture 22 having one or moreaperture engagement features 42, such as threads. In addition, the cap12 may include a blocking member 46 which may have one or more blockingmember engagement features 44, such as threads, for engaging theaperture engagement features 42. For example, the blocking member 46 maybe removably coupled to the aperture 22 such that when the blockingmember is secured to the aperture, one or more fluids or materials, maybe contained within the interior space 20 of the cap. However, upondecoupling of the blocking member 46 to the aperture 22, the one or morefluids or materials may be released from the interior space 20 in thecap 12. For example, once the cap 12 has at least been partially securedto the tube 14, the blocking member 46 may be decoupled from theaperture 22, thereafter allowing fluids or materials in the interiorspace 20 to be released into the reservoir 40 of the tube 14. The one ormore fluids or materials contained in the interior space 20 in the cap12 may assist in preserving the sample body fluids contained in thereservoir 40 of the tube 14 during at least storage and shipping. Any ofthe engagement features discussed herein may be any number of engagementfeatures for allowing temporary or permanent engagement between twoparts or features of the sample collection device 10 and are not limitedto the examples discussed in this disclosure.

The blocking member 46 may also include one or more coupling features 48which may allow one or more coupling members 50 comprising a part of thetube 14 to engage and couple with the coupling features 48. The couplingbetween the coupling features 48 and coupling members 50 can assist indecoupling the blocking member 46 from the aperture 22. For example, asthe cap 12 is secured to the tube 14, the coupling member 50 may engageand interact with the coupling feature 48 of the blocking member 46,such as similar to the head of a screw driver interacting with the headof a screw. The blocking member 46 may be threadably engaged withthreaded aperture engagement features, and the coupling and interactionof the coupling feature 48 and coupling member 50 may cause the threadedengagement between the blocking member 46 and the aperture 22 to bereleased. The threaded engagement between the blocking member 46 and theaperture 22 may be released, for example, due to rotation of theblocking member 46 relative to the aperture 22. Any number of releasableengagements may be used to engage the blocking member 46 with theaperture 22 such that the engagement between the blocking member 46 andthe aperture 22 may be released upon securing the cap to the tube 14.Similarly, any number of features may be integrated in the samplecollection device 10 which may allow containment of a solution in a partof the cap 12 or tube 14 such that the solution is not released untilthe cap is at least partially secured to the tube 14.

The tube 14 in FIG. 1A is shown by way of example as having a couplingmember 50 in the shape of a square peg which is complementary to asquare shaped indent comprising the coupling feature 48 in the blockingmember 46. Furthermore, the coupling member 50 can be centrally locatedwithin the tube 14 and the coupling feature may be centrally located onthe bottom of the blocking member 46. Therefore, upon threadedengagement between the cap 12 and the tube 14, the square peg couplingmember 50 may extend into and engage the square shaped indent couplingfeature 48 in the blocking member 46, thus preventing the blockingmember 46 from rotating relative to the coupling member 50. However,although the blocking member 46 may be prevented from rotating relativeto the coupling member 50, the blocking member 46 may rotate relative tothe aperture 22 and become disengaged from the aperture 22, such as fromreleasing the threaded engagement between the blocking member 46 andaperture 22. FIG. 1B shows an example coupling member 50 secured to aninner surface 52 of the containment wall of the tube 14 by more than onecross-member 54. The one or more cross members 54 can assist in securingthe position of the coupling member 50 while allowing space for thepassage of fluids or materials into the reservoir 40.

An example method of use of a sample collection device 10 can includethe sample collection device 10 supplied with sample preservation fluidin the interior space 20 of the cap 12, and with the blocking member 46threadably engaged with the aperture 22 in order to contain the samplepreservation fluid in the interior space 20. Sample fluid, such assaliva or urine, may then be placed in the reservoir 40 of the tube 14by a donor. The cap 12 can then be screwed onto the tube 14. Screwingthe cap 12 onto the tube 14 may cause the coupling member 50 in the tube14 to engage the coupling feature 48 of the blocking member 46 andunscrew the blocking member 48 from the aperture 22 and into theinterior space 20 of the cap 12. Decoupling the blocking member 48 fromthe aperture 22 can allow the sample preservation fluid to flow into thereservoir 40 of the tube 40. After release of the sample preservationfluid into the reservoir 40 of the tube 14, the sample preservationfluid can mix with the donor's sample fluid, thereby preserving thedonor's sample fluid.

While shown as a square peg in this illustration, the coupling member 50of the tube 14 can be any shape that is complementary in shape with thecoupling feature 48 of the blocking member 46 such that it allows theblocking member 46 to decouple from the aperture 22. The couplingfeature 48 can be either in the blocking member 46 or the tube 14, andthe complimentary coupling member 50 may be either in the tube 14 orblocking member 46, respectively. Other shapes will be evident to oneskilled in the art, including, without limitation, a slot and a tab,like a regular screwdriver and screw, or a cross-shaped pair, like aPhillips screwdriver and screw.

An additional embodiment of the sample collection device 100 is shown byway of example in FIGS. 2A and 2B. The sample collection device 100 mayinclude one or more coupling members 50 and complimentary couplingfeature 48 which may be placed eccentrically from either the cap 12 ortube 14. As shown in FIG. 2B, less material and parts may be requiredfor this embodiment to work properly, such as the coupling member 50maintaining proper positioning by only one cross-member 54. Although thecoupling member 50 is shown as being held in position by only onecross-member 54 extending from the containment wall 32 of the tube 14,any number of configurations and cross-members 54 may be used toposition the coupling member 50 without departing from the scope of thisdisclosure.

Another embodiment of the sample collection device 200 is shown by wayof example in FIGS. 3A-3C. More specifically, FIG. 3A shows a crosssection of the cap 12 prior to being coupled to the tube 14. The cap 12can include an outer wall 24 and cap engaging members 34 along an insidesurface 26 of the outer wall 24. The interior space 20 may be at leastpartially defined by at least one of an inner wall 18 or outer wall 24of the cap 12. Furthermore, the inner wall 18 can include engagementfeatures 60, such as threads, along a surface of the inner wall 18. Theinner wall 18 may further define an aperture 22 which may be open orclosed depending on the position of an annular blocking member 62relative to the aperture 22. When the aperture 22 is closed such thatthe annular blocking member 62 is covering the aperture 22, fluid ormaterial, such as sample preservation fluid or material 70, that me becontained in the interior space 20 may not be allowed to travel outsideof the interior space 20, as shown in FIG. 3A. However, when theaperture 22 is open such that the annular blocking member 62 is notcovering the aperture 22, the fluid or material 70 that may be containedin the interior space 20 may be allowed to travel outside of theinterior space 20, such as into the reservoir 40 of the tube 14, asshown in FIG. 3B. The fluid or material 70 contained in the interiorspace may be beneficial for preserving sample 72, such as body fluids(i.e., saliva, urine, etc.) placed in the reservoir 40 of the tube 14,similarly as described above. Furthermore, any number of mechanisms mayprevent the sample preserving fluid or material 72 from being releasedfrom the interior space 20 until the cap 12 is at least partiallysecured to the tube 14.

In the embodiment shown by way of example in FIGS. 3A-3C, the annularblocking member 62 may be configured to interact with one or morefeatures, such as a coupling member 50, of the tube 14 such that as thecap 12 is being securely coupled to the tube 14, the one or morefeatures of either the tube 14 or annular blocking member 62 can causethe annular blocking member 62 to move from a position where the annularblocking member 62 is covering the aperture 22 to a position where theannular blocking member 62 is not covering the aperture 22, thusallowing the sample preserving fluid or material 72 to release from theinterior space 20 and interact with the sample 72.

FIG. 3C shows a cross section of the tube 14, having a containment wall32 defining a reservoir 40 for sample collection. The tube 14 caninclude a coupling member 50 for engaging the coupling feature 48 of theannular blocking member 62.

An example method of use of a sample collection device 200 can includethe sample collection device 200 supplied with sample preservation fluid70 in the interior space 20 of the cap 12, and with the annular blockingmember 62 covering the aperture 22 in order to prevent the passage ofsample preservation fluid 70 through the aperture 22. In thisembodiment, sample fluid 72, such as saliva or urine, can be placed inthe reservoir 40 of the tube 14. The cap 12 may then be securelycoupled, such as threadably engaged, onto the tube 14 causing thecoupling features 48 of the annular blocking member 62 to engage thecoupling member 50 of the tube 14. The annular blocking member 62 canthen threadably engage the engagement features, such as threads, alongthe side of the inner walls. This can cause the annular blocking member62 to move away from the aperture 22 so that it no longer covers theaperture 22. This, in turn, can release at least some of the samplepreservation fluid 70 into the reservoir 40 of the tube 14, where it canmix with the sample fluid 72, thereby preserving it.

In some embodiments, the sample collection device 300, as shown by wayof example in FIGS. 4A and 4B, cap 12 includes at least one couplingfeature or a wedge 90 which is shaped and configured to interact with acomplimenting coupling feature or a flange 92 of the tube 14. In thisembodiment, the wedge 90 and flange 92 are extending along an insidesurface of the cap 12 and tube 14. For example, when the cap 12 iscoupled to the tube 14, the wedge 90 can engage the flange 92 and form asecure engagement between the cap 12 and the tube 14. Furthermore, oncethe wedge 90 and flange 92 have been completely engaged with each other,such as the locked configuration 96 shown by way of example in FIG. 4B,the engagement between the wedge 90 and the flange 92 may not bereleasable by at least the sample donor. Therefore, once the cap 12becomes engaged to the tube 14 such that the wedge 90 and flange 92 aresecurely engaged with each other, the cap 12 may no longer be disengagedfrom the tube 14 by at least the sample donor. This can prevent at leastthe sample donor from contaminating the sample body fluid that wasdeposited in the tube 14, as well as protect the sample donor fromcontact with the sample preservation solution. FIG. 4B shows sampleembodiments of the unlocked configuration 94 and locked configuration 96between the wedge 90 and flange 92.

In some embodiments, the sample collection device 400, as shown by wayof example in FIGS. 5A and 5B, the wedge 90 and flange 92 are extendingalong an outside surface of the cap 12 and tube 14, respectively. FIG.5B shows sample embodiments of the unlocked configuration 94 and lockedconfiguration 96 between the wedge 90 and flange 92.

In some embodiments, the sample collection device 500, as shown by wayof example in FIG. 6, where the cap 12 includes one or more sealedcavities 110 containing a sealing substance 112, such as glue. Any onesealed cavity 110 may be either operatively associated or positionedadjacent engagement features 34, such as threads, on the cap 12 suchthat when the cap 12 is coupled to the tube 14 the one or more sealedcavities 110 may be broken by one or more features or end 150 of thetube 12. Once the sealed cavity 110 is broken, a sealing substance 112,such as glue, may be released from the sealed cavity 110 and cause thecap 12 to become permanently secured to the tube 14.

Any number of features may be included with the cap 12 or tube 14 whichmay assist in preventing unwanted decoupling of the cap 12 from the tube14, such as to prevent contamination. Additionally or alternatively,either the cap 12 or tube 14 may include a “tamper evident” feature 160which may become altered such that it can be known to a user or samplecollector if the cap 12 has been unfavorably decoupled from the tube 14.As shown by way of example in FIGS. 7A and 7B, the cap 12 may include atamper evident feature 160 which may be comprised of a ring that isreleasably attached to the open end 162 of the cap 12 such that when thecap 12 is unfavorably decoupled from the tube 14, the tamper evidentfeature 160 can permanently release its attachment from the cap 12, asshown in FIG. 7B. Once the tamper evident feature 160 is permanentlydetached from the cap 12, any observer of the cap 12 can determine thatthe cap 12 had been unfavorably decoupled from the tube 12, thusproviding a warning of sample contamination, for example.

Those skilled in the art will recognize that numerous equivalentembodiments can be used to obtain the benefits provided by the samplecollection devices disclosed herein. For example, while thisspecification refers to certain elements being in the cap 12, and othersin the tube 14, one skilled in the art would recognize that reversingthe elements in the cap 12 to be in the tube 14 and vice-versa, would bean equivalent.

In some embodiments, a solution for preserving cells in one or morebodily fluids, such as saliva and urine, is disclosed. The solution forpreserving cells may be beneficial for further separation into celltypes and downstream molecular analysis that allows for storage of cellsin the body fluid to retain their antigenicity and cellulararchitecture. The solution may contain at least one chemical fixingagent, such as but not limited to paraformaldehyde, and at least oneprotease inhibitor. In some embodiments, the solution may furthercontain one or more of at least one antimicrobial agent, and serumproteins from human and/or other animal species. The solution can bebuffered at a pH from between about 6.4 to about 8.4, preferably frombetween about 7.2 to about 7.6.

For purposes of the disclosure, “preserving cells” means preventing thecells from having their antigens degraded, such that they can bepurified or enriched based on their antigens, and preventing alterationsin the cellular epigenome. The “epigenome” means the state or pattern ofalteration of genomic DNA by covalent modification of the DNA or ofproteins bound to the DNA. Examples of such alteration includemethylation at the 5 position of cytosine in a CpG dinucleotide,acetylation of lysine residues of histones, and other heritable ornon-heritable changes that do not result from changes in the underlyingDNA sequence.

In some embodiments, concentrations of agents in the followingdescription can be those of the sample preserving solution itself.Depending upon the bodily fluid, and in the case of saliva, about anequal volume of solution and body fluid can be mixed together. Thispreferably results in the cells from the body fluids retaining theirantigenicity and DNA integrity for at least one week at roomtemperature.

In some embodiments of the disclosure, the volume of preservationsolution held within the device and deployed may be between about 100and about 500 ml, which is relevant, for example, for the preservationof cells in urine. As such, the preservation solution for urine may beanywhere between about ten times (10×) concentrated solution to aone-point five time (1.5×) solution for urine.

A “chemical fixing agent”, according to some embodiments, is a chemicalcross-linking compound used to alter cell components such that the cellsresist degradation. The chemical fixing agents can also serve tocross-link histones and other DNA-binding proteins to the DNA. Suchagents may be known in the art and include, without limitation,paraformaldehyde, formaldehyde, formalin, aldehydes, alcohol, oxidizingagents, Mercurials, Picrates, Hepes-glutamic acid buffer-mediatedorganic solvent protection effect (HOPE), fixative combinations such asZambonis fixative, combinations of aldehydes, and syntheticcross-linking reagents. In some embodiments, the chemical fixing agentis paraformaldehyde. In some embodiments, the chemical fixing agent ispresent at a concentration of about 1% (v/v).

To protect the cells from degradation by proteases present in the bodyfluids, in some embodiments, the solution can contain at least oneprotease inhibitor. In some embodiments, the protease inhibitor can beselected from the group consisting of Aspartic protease inhibitors,Cysteine protease inhibitors, Metalloprotease inhibitors, Serineprotease inhibitors (e.g., serpins), Threonine protease inhibitors,Trypsin inhibitors, and Kunitz STI protease inhibitor. Some specific,non-limiting, examples include sodium azide, PMSF, Aprotinin, leupeptin,pepstatin, natural or synthetic proteinase inhibitors, and cocktailmixtures of protease inhibitors. Suitable concentrations of theseinhibitors can include, without limitation, PMSF (Phenylmethylsulfonylfluoride) Serine proteases at about 0.1-1 mM, Benzamidine Serineproteases at about 1 mM, Pepstatin A Acid proteases at about 1 μg/ml,Leupeptin Thiol proteases at about 1 μg/ml, Aprotinin Serine proteasesat about 5 μg/ml, and Antipain Thiol proteases at about 1 μg/ml. Incertain embodiments, the protease inhibitor is sodium azide at aconcentration of about 0.01% (w/v).

To prevent damage to the cells from microbial contamination, someembodiments of the solution contain at least one antimicrobial agent.Suitable antimicrobial agents include, without limitation, antibacterialand antifungal antibiotics.

Preservation of cell architecture is enhanced by the presence of serumproteins, which may optionally be added to the solution in someembodiments. Additionally serum proteins may be used to neutralizeosmotic difference between cells and solution. These can be from humanor other animal sources. In some cases, whole serum may be used. Forexample, fetal bovine serum may be added, in some embodiments at about1% (v/v).

The solution according to the disclosure may include any combination ofthe foregoing embodiments.

In some embodiments of the disclosure, a method for preserving cells inone or more bodily fluids is disclosed. The method for preserving thecells can comprise contacting the body fluids with the solutionaccording to the present disclosure. The body fluids can contain avariety of cell types and the cells in the body fluids can be preservedby the solution according to the present disclosure. While not criticalto the present disclosure, a ratio of solution to body fluids of fromabout 1 to 1 is typically used.

The following examples are intended to further illustrate someembodiments of the solutions and methods for preserving cells in bodyfluids and are not to be construed to limit the scope of thisdisclosure.

For example, a solution of PBS pH 7.4, 1% Paraformaldehyde, 1% FBS, and0.01% NaN3 can be added at a 1:1 ration with saliva, then T-cells can bepurified and DNA extracted. The results of such a process are shown inFIG. 8. These results can demonstrate that the integrity of theantigenicity and DNA of T-cells was maintained for at least one week.

In some embodiments of the present disclosure, a method is disclosedwhich provides a sample of one or more body fluids, such as saliva orurine, comprising chemically fixed cells, and optionally centrifugingthe body fluid sample to separate DNA and other soluble material from apellet of cells including bacteria and debris. The method can furtherinclude enriching white blood cells, including lymphocyte cells, fromother contents of the pellet. Additionally, specific cells may beisolated using antibodies conjugated to magnetic beads targeted to cellspecific markers.

In some embodiments, the disclosure provides a method for isolating aparticular type of white blood cell, specifically including, but notlimited to lymphocytes, from bodily fluids (i.e., saliva, urine, etc.),comprising, for example one or more (and in some embodiments, several orall of the steps): providing a body fluid sample comprising chemicallyfixed cells, optionally centrifuging the body fluid sample to obtain apellet comprising cells, optionally resuspending the pellet in buffer,subjecting the re-supended pellet to density gradient separation toobtain a layer of a mixture of white blood cell types (includinglymphocytes), contacting the mixture of cell types with a solutioncontaining specific binding agents for an epitope found on a particulartype of white blood cell, and separating the particular type of whiteblood cell (including lymphocytes) from the mixture of white blood celltypes.

In some embodiments, the specific binding agents can include magneticbeads coupled to antibodies specific to an epitope found on a particulartype of white blood cell, and separating may comprise magneticallyseparating the particular type of white blood cell (includinglymphocytes) from the mixture of white blood cell types, though anymethod (and corresponding system/device) for separating cell types fromone another is within the scope of this disclosure. Magnetic separationis but one method for doing so.

The cells can be chemically fixed prior to being subjected to the methodaccording to this disclosure. The cells can be chemically fixed by,e.g., contacting a sample of saliva with a chemical fixation solution.This is done to preserve the cells over time at ambient temperatures.This can also allow for a complete study of the epigenome as it allowshistone modifications and other protein-DNA interactions to be studiedfrom the deposited body fluid samples. Histones must be chemically fixedto the DNA in order to be studied. Without fixation, the histonesgenerally cannot remain bound to the DNA and the proteins can degradeover time.

In some embodiments, the buffer can comprise sodium azide, the buffercan comprise phosphate buffered saline and sodium azide, In someembodiments, the buffer may further comprise fetal bovine serum. In someembodiments, the buffer is at a pH from between about 7.2 to about 7.6.

In some embodiments, the cells are washed once in buffer. This inpractice removes soluble material and in the case of saliva it removeswhat has been classified as the “buccal” layer (Dos-Santos et al.,2009).

In some embodiments, the mixture of white blood cells is washed one ormore times in buffer prior to separating. This is preferably done toremove any remaining density gradient solution from the mixture of celltypes.

In the process, the antibodies may bind to the particular type of whiteblood cells, thus binding the particular type of white blood cells tothe magnetic beads. The particular type of white blood cells can then beseparated from any other cell types by placing the magnetic beads in amagnetic field and removing any remaining liquid to obtain isolatedcells of the particular type of white blood cells.

In some embodiments, the particular type of white blood cells can be alymphocyte, where the lymphocyte may be a T-cell. In such embodiments,the antibodies used may be specific to an antigen specific to T-cells(e.g., the antigen being CD4). In some embodiments, the isolated bloodcells may then be frozen prior to further processing, such as prior toepigenetic analysis.

The following example is intended to further illustrate an examplemethod embodiment of the present disclosure and is not intended to limitthe scope of the disclosure.

Example: Isolating T-Cells from a Bodily Fluid (e.g., Saliva)

Saliva is collected, and the saliva is mixed with preservation solution.The cells are then pelleted by centrifugation and the processingsolution is removed. The cells are then re-suspended in about 6 mlbuffer (PBS, pH 7.4), 1% FBS, 0.01% NaN3), then washed once in a bufferand repelletted. The pellet are resuspended in about 6 mL PBS-15FBS-.01% NaN3 and subjected to density gradient centrifugation using1.082-1.072 g/ml of Ficoll® (GE Healthcare). The white-blood cells arespun to the interface of the polysaccharides and buffer while thebacteria, debris, and any other particulate matter were pelleted at thebottom of the tube. The cells are extracted from the tube and placed ina new tube. The cells are then washed in Hank's Balanced Salt Solutiononce and then washed with the PBS-NaN3-FBS buffer once to removeremaining density gradient solution that may have been taken whileextracting the white blood cells from the interface.

The sample now includes highly enriched white-blood cells with minimalbacteria and minimal debris. This step can also greatly decrease othercell types, such as epithelial cells. The cells can then be incubated inbuffer (PBS-NaN3-FBS) with antibody targeted against CD4 conjugated tomagnetic beads (Dynabeads® Invitrogen®). The samples can then be placedin a magnetic field, the beads brought to the side of the tube, and theliquid removed. The liquid may contain everything not bound to the beadsthrough the antibody. The T-cells can be bound to the antibody and notremoved due to the magnetic field. The beads and the attached cells canbe washed in buffer to eliminate any non-specific or weak binding ofother cells, bacteria, or other debris found in bodily fluids, such assaliva or urine. The cells can then be frozen for later downstreamprocessing and analysis. The isolation of T-cells can be confirmed bylight microscopy (T-cells are very distinct compared to epithelial cellsand bacteria) (see FIG. 9). Additionally, flow cytometry and F.A.C.S.analysis using antibodies against CD3, CD4, and CD8 can confirm visualassessment of the isolated cells. The T-cells may then be tittered fromthe body fluid to determine the number of T-cells per unit of body fluid(ml) in order to determine the amount of body fluid, such as saliva orurine, for an adequate number of cells for downstream experimentation(see FIGS. 9 and 10). The isolated cells can be shown to have DNA devoidof degradation and appropriate for downstream use (see FIG. 8).

Any and all references to publications or other documents, including butnot limited to, patents, patent applications, articles, webpages, books,etc., presented in the present application, are herein incorporated byreference in their entirety.

Although a few variations have been described in detail above, othermodifications are possible. For example, any logic flow depicted in theaccompanying figures and described herein does not require theparticular order shown, or sequential order, to achieve desirableresults. Other implementations may be within the scope of at least someof the following exemplary claims.

Example embodiments of the devices, systems and methods have beendescribed herein. As noted elsewhere, these embodiments have beendescribed for illustrative purposes only and are not limiting. Otherembodiments are possible and are covered by the disclosure, which willbe apparent from the teachings contained herein. Thus, the breadth andscope of the disclosure should not be limited by any of theabove-described embodiments but should be defined only in accordancewith claims supported by the present disclosure and their equivalents.Moreover, embodiments of the subject disclosure may include methods,systems and devices which may further include any and all elements fromany other disclosed methods, systems, and devices, including any and allelements corresponding to collection, preservation, separating andisolating of cells from bodily fluids (e.g., saliva, urine), as well asthe collection of other substances, including toxic and/or hazardoussubstances/fluids (as well as the preservation, separating and isolationof components thereof). In other words, elements from one or anotherdisclosed embodiments may be interchangeable with elements from otherdisclosed embodiments.

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What is claimed is:
 1. A biological sample collection system, the systemcomprising: a sample collection vessel, the sample collection vesselcomprising a sample collection reservoir having an opening configured toreceive a biological sample into the sample collection reservoir; amovable annular valve configured to associate with the opening of thesample collection reservoir; and a cap, the cap comprising a reagentchamber configured to store a reagent therein and configured toassociate with the movable annular valve and with the sample collectionvessel, wherein associating the cap with the sample collection vessel isconfigured to cause the movable annular valve to open, therebydispensing the reagent into the sample collection reservoir.
 2. Thebiological sample collection system of claim 1, wherein the samplecollection vessel further comprises a connection member.
 3. Thebiological sample collection system of claim 2, wherein the cap furthercomprises a complementary connection member configured to associate withthe connection member of the sample collection vessel to couple thesample collection vessel and the cap.
 4. The biological samplecollection system of claim 3, wherein the connection member comprisesone of a pushed connection, a rotated connection, a screwed connection,and a snap-fit connection.
 5. The biological sample collection system ofclaim 3, wherein the connection member and the complementary connectionmember each comprise threads.
 6. The biological sample collection systemof claim 5, wherein the threads of the complementary connection membercomprise a plurality of internal threads of the cap.
 7. The biologicalsample collection system of claim 1, wherein the sample collectionsystem comprises a separable two-piece sample collection system, thesample collection vessel comprising a first piece of the separabletwo-piece sample collection system, and the movable annular valveassociated with the cap comprising a second piece of the separabletwo-piece sample collection system.
 8. The biological sample collectionsystem of claim 1, wherein the movable annular valve comprises an innercylinder and an outer cylinder.
 9. The biological sample collectionsystem of claim 8, wherein the inner cylinder comprises at least onefluid vent.
 10. The biological sample collection system of claim 9,wherein an interior sidewall of the outer cylinder defines an apertureconfigured to accommodate at least a portion of the inner cylinder. 11.The biological sample collection system of claim 10, wherein the atleast one fluid vent is configured to be obstructed by the outercylinder when the movable annular valve is closed, and wherein the atleast one fluid vent is configured to be unobstructed by the outercylinder when the movable annular valve is open.
 12. The biologicalsample collection system of claim 8, wherein the outer cylindercomprises threads on an interior sidewall configured to mate withthreads on an exterior sidewall of the inner cylinder.
 13. Thebiological sample collection system of claim 8, wherein the innercylinder associates with the cap to create a fluid-tight seal.
 14. Thebiological sample collection system of claim 13, wherein the innercylinder additionally comprises a reagent retention chamber, and whereinthe reagent retention chamber is in fluid communication with the reagentchamber of the cap when the inner vessel is associated with the cap. 15.The biological sample collection system of claim 8, wherein the outercylinder comprises a sample collection reservoir sealing surfaceconfigured to create a fluid-tight seal between the outer cylinder andthe opening of the sample collection reservoir when the outer cylinderis associated with the sample collection reservoir.
 16. The biologicalsample collection system of claim 15, wherein a diameter of an outerwall of the inner cylinder is substantially equal to a diameter of theinner wall of the outer cylinder such that when the outer cylinder isassociated with the inner cylinder, a fluid-tight connection is createdtherebetween.
 17. A method for collecting and preserving a biologicalsample, the method comprising: receiving a biological sample at a samplecollection vessel; and associating a cap with the sample collectionvessel that is configured to cause a movable annular valve associatedwith the cap to open, thereby releasing a reagent stored within the capinto the sample collection reservoir.
 18. The method of claim 17,wherein receiving a biological sample at the sample collection vesselcomprises receiving a biological sample through an opening of a samplecollection reservoir within the sample collection vessel.
 19. The methodof claim 17, wherein associating the cap with the sample collectionvessel comprises threadedly engaging a connection member disposed on anexterior surface of the sample collection vessel with a complementaryconnection member disposed on an interior surface of the cap.
 20. Themethod of claim 17, wherein associating the cap with the samplecollection vessel that is configured to cause the movable annular valveassociated with the cap to open comprises moving a distal portion of aninner cylinder through an aperture defined by an interior sidewall of anouter cylinder to expose a fluid vent defined by the distal portion ofthe inner cylinder, the inner cylinder and the outer cylinder comprisingthe movable annular valve.
 21. The method of claim 20, wherein the outercylinder comprises threads on the interior sidewall configured to matewith threads on an exterior sidewall of the inner cylinder, and whereinmoving a distal portion of the inner cylinder through the aperturecomprises uncoupling the respective threads.
 22. The method of claim 20,wherein the inner cylinder associates with the cap to create afluid-tight seal.
 23. The method of claim 20, wherein the outer cylindercomprises a sample collection reservoir sealing surface configured tocreate a fluid-tight seal between the outer cylinder and the opening ofthe sample collection reservoir when the outer cylinder is associatedwith the sample collection reservoir.
 24. The method of claim 17,further comprising at least partially disassociating the cap from thesample collection vessel to cause the movable annular valve associatedwith the cap to close.
 25. A kit for collecting and preserving abiological sample, the kit comprising: a sample collection vessel, thesample collection vessel comprising: a sample collection reservoirhaving an opening configured to receive the biological sample from auser into the sample collection reservoir; a connection member disposedon an exterior portion the sample collection vessel and adjacent to theopening; a cap, the cap comprising: a reagent chamber configured tostore a reagent; and a complementary connection member configured toengage the connection member of the sample collection vessel; and amovable annular valve configured to associate with the cap and with theopening of the sample collection reservoir.
 26. The kit of claim 25,wherein the movable annular valve comprises an inner cylinder, the innercylinder being in fluid-tight association with the cap.
 27. The kit ofclaim 26, wherein the movable annular valve further comprises an outercylinder, the outer cylinder configured to be in fluid-tight associationwith the inner vessel and configured to associate with the opening ofthe sample collection reservoir.
 28. The kit of claim 27, wherein theouter cylinder comprises threads on the interior sidewall configured tomate with threads on an exterior sidewall of the inner cylinder, andwherein moving a distal portion of the inner cylinder through theaperture comprises unscrewing the annular valve with the respectivethreads.
 29. The kit of claim 27, wherein the outer cylinder comprises asample collection reservoir sealing surface configured to create afluid-tight seal between the outer cylinder and the opening of thesample collection reservoir when the outer cylinder is associated withthe sample collection reservoir.
 30. The kit of claim 25, furthercomprising a funnel configured to associate with the sample collectionvessel and to guide receipt of a biological sample from a user into thesample collection reservoir of the sample collection vessel.